Diagnosing Neurodegenerative Diseases by Label-Free 3-D Imaging of Intestinal Samples

Early diagnosis of Parkinsons disease (PD) remains challenging because motor symptoms appear only after extensive neurodegeneration, and a definitive diagnosis still relies on post-mortem neuropathology. Increasing evidence implicates the enteric nervous system (ENS) in prodromal disease stages, but routine ENS-based diagnosis is limited by the complexity of intestinal tissue organization and the need for specific labeling strategies. Here, we developed a label-free autofluorescence (AF) imaging workflow combined with unbiased morphometric analysis to identify neurodegenerative alterations in fixed human colonic tissue. Using a correlative multiscale imaging approach, we generated a database of almost 800 high-resolution confocal images from myenteric and submucosal plexuses of controls, PD, and Alzheimers disease (AD) patients. Blind evaluation by four expert histologists showed reliable identification of control tissue but lower sensitivity for pathological cases, reflecting the heterogeneous distribution of ENS lesions. Semi-quantitative and morphometric image analyses identified a distinct population of enlarged enteric neurons, termed large neural cells (LNCs), strongly enriched in PD and AD compared with controls. LNCs contained autofluorescent cytoplasmic inclusions and frequently prominent nucleoli, both features largely absent from control tissue independent of aging. Co-localization with the amyloid-binding probe Amytracker (AmyT) demonstrated that AF granules correspond to {beta}-sheet-rich protein aggregates rather than merely age-related lipofuscin granules. Similar alterations were detected in intact three-dimensional (3-D) colonic biopsies, demonstrating the feasibility of volumetric ENS imaging without tissue clearing. Together, our results establish label-free AF imaging as a rapid and clinically compatible strategy for detecting enteric neurodegenerative pathology. This approach provides a framework for the future development of ENS-based biomarkers and supports the use of volumetric intestinal imaging for early diagnosis of neurodegenerative diseases.